This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To target the Wisconsin National Primate Research Center's mission to develop treatments for human disease, generate knowledge of primate biology, and to facilitate the progress of research by providing expertise and resources to external scientists, we are developing a methodology that we will use to determine the abundance and in situ localization of CD4 T cells specific to simian immunodeficiency virus (SIV) in tissues during SIV infection. Results: We initiated these studies by working to develop methods to stain antigen specific CD4 T cells in mice. In collaboration with Dr. Marc Jenkins and Dr. T. Dileepan at the University of Minnesota we are using mice infected with bacteria that are engineered to express a peptide that triggers a peptide-specific CD4 T cell response for which MHC class II tetramers are available. We are staining nasal associated lymphoid tissues (NALT) and spleen tissues because peptide-specific CD4 T cells accumulate at these sites. We have tried staining tissues with class II tetramers at several different concentrations, at several different lengths of incubation time, at different temperatures, and using several amplification techniques. Thus far, we have been successful at detecting cells that are weakly stained with tetramers in the NALT using 2-4 nM tetramers incubated over night at four degrees followed by a one hour incubation at room temperature. We are now attempting to improve the detection of cells with the class II tetramers using tyramide signal amplification (TSA) techniques, and verifying the specificity of staining using CD4, CD3, and CD20 antibody counterstaining. In addition, Dr. Nancy Wilson at the WNPRC is making progress developing class II reagents for use in SIV-infected macaques. She has thus far successfully produced rhesus macaque class II molecules and is now working to develop a method to purify these molecules. In addition, 9 more DP/peptide trimers have been identified, as have 4 more DQ trimers and 10 more DR trimers. Work will progress on these subsequent to the successful completion of the seven tetramers we are studying. This work used WNPRC Research Services. PUBLICATIONS: *Jung Joo Hong, Teresa L. Mattila, Aaron Hage, Matthew R. Reynolds, David I. Watkins, Christopher J. Miller, and Pamela J. Skinner, Localized Populations of CD8- SIV-Specific T Cells in Lymphoid Follicles and Vaginal Epithelium of SIV infected macaques. PloS ONE, 2009;4(1): e4131. Qingsheng Li, Pamela J. Skinner, Sang-Jun Ha, Lijie Duan, Teresa L. Mattila, Aaron Hage, Cara White, Daniel L. Barber, Leigh O'Mara, Peter J. Southern, Cavan S. Reilly, Christopher J. Miller, Rafi Ahmed and Ashley T. Haase, Visualizing antigen specific and infected cells in situ predicts outcome in early viral infection. Science, 2009;323(5922):1726-1729. Qingsheng Li, Pamela J. Skinner, Lijie Duan, and Ashley T. Haase, A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells in situ, published in both Science and Journal of Visualized Experimentation, 2009.